Spectral domain phase microscopy (SDPM) dual mode imaging systems and related methods and computer program products

ABSTRACT

Some embodiments of the present invention provide optical coherence tomography systems including an OCT engine and a processor. The OCT engine is configured to provide both standard OCT imaging and spectral domain phase microscopy (SDPM) imaging. The processor is coupled to the OCT engine and is configured to use a first signal processing method when the OCT engine is configured to provide standard OCT imaging and a second signal processing method when the OCT engine is configured to provide SDPM imaging. Related methods and computer program products are also provided.

CLAIM OF PRIORITY

The present application claims priority from U.S. ProvisionalApplication No. 60/736,945, filed Nov. 15, 2005, the disclosure of whichis hereby incorporated herein by reference as if set forth in itsentirety.

FIELD OF THE INVENTION

The present invention relates to imaging and, more particularly, tooptical coherence imaging devices, methods and systems.

BACKGROUND OF THE INVENTION

Since its introduction in the early 1990's, optical coherence tomography(OCT) has emerged as a promising imaging modality for micrometer-scalenoninvasive imaging in biological and biomedical applications. Itsrelatively low cost and real-time, in vivo capabilities have fueled theinvestigation of this technique for applications in retinal and anteriorsegment imaging in opthalmology (e.g., to detect retinal pathologies),early cancer detection and staging in the skin, gastrointestinal, andgenitourinary tracts, as well as for ultra-high resolution imaging ofentire animals in embryology and developmental biology.

Conventional OCT systems are essentially range-gated low-coherenceinterferometers that have been configured for characterization of thescattering properties of biological and other samples. By measuringsingly backscattered light as a function of depth, OCT fills a valuableniche in imaging of tissue ultrastructure, and provides subsurfaceimaging with high spatial resolution (˜1-10 μm) in three dimensions andhigh sensitivity (>110 dB) in vivo with no contact needed between theprobe and the tissue. OCT is based on the one-dimensional technique ofoptical coherence domain reflectometry (OCDR), also called opticallow-coherence reflectometry (OLCR). See Youngquist, R. C., S. Carr, andD. E. N. Davies, Optical Coherence Domain Reflectometry: A New OpticalEvaluation Technique. Opt. Lett., 1987. 12: p. 158; Takada, K., et al.,New measurement system for fault location in optical wave guide devicesbased on an interferometric technique. Applied Optics, 1987. 26(9): p.1603-1606; and Danielson, B. L. and C. D. Whittenberg, Guided-waveReflectometry with Micrometer Resolution. Applied Optics, 1987. 26(14):p. 2836-2842. In some instances of time-domain OCT, depth in the sampleis gated by low coherence interferometry. The sample is placed in thesample arm of a Michelson interferometer, and a scanning optical delayline is located in the reference arm.

The time-domain approach used in conventional OCT has been used insupporting biological and medical applications. An alternate approachinvolves acquiring as a function of optical wavenumber theinterferometric signal generated by mixing sample light with referencelight at a fixed group delay. Two methods have been developed whichemploy this Fourier domain (FD) approach. The first is generallyreferred to as Spectral-domain OCT (SD-OCT). SD-OCT uses a broadbandlight source and achieves spectral discrimination with a dispersivespectrometer in the detector arm. The second is generally referred to asswept-source OCT (SS-OCT). SS-OCT time-encodes wavenumber by rapidlytuning a narrowband source through a broad optical bandwidth. Both ofthese techniques can provide improvements in SNR of up to 15-20 dB whencompared to time-domain OCT, because SD-OCT and SS-OCT capture thecomplex reflectivity profile (the magnitude of which is generallyreferred to as the “A-scan” data or depth-resolved sample reflectivityprofile) in parallel. This is in contrast to time-domain OCT, wheredestructive interference is employed to isolate the interferometricsignal from only one depth at a time as the reference delay is scanned.

However, the resolution of current OCT techniques is generally limitedby the coherence length of the illumination source. Therefore, currentOCT techniques may not be able to resolve structures of less than ˜1-10μm. For example, the characteristics and dynamics of the cellularsurface may be of interest in many areas of quantitative biology.However, there are few scientific tools which are capable ofnoninvasively acquiring quantitative information about cell surfaceprofiles, displacements, and motions on the nanometer scale.

Recent advances have increased the imaging speed, which may allowrelatively large image sets (such as 3-D volumes) to be quicklygenerated. Since OCT is high-speed, non-contact and non-destructive, itis well suited for imaging dynamics over short time scales, for example,well below 1 second (the beating of a heart tube in a fruit fly) all theway up to changes over a long time scales, for example, days or evenlonger (tissue growing).

Spectral domain phase microscopy (SDPM) is an extension of SD-OCT(38-40) which may allow for the measurement of nanometer-scaledisplacements and motions within each pixel of an SD-OCT image. Themajor modifications in SDPM as compared to SD-OCT are the substitutionof a common path interferometer for the Michelson interferometer inSD-OCT, and enhanced signal processing based on the measured phase ofthe SD-OCT signal in each image pixel to extract nanometer-scaledisplacement and motion of reflectors and scatterers located within thatpixel. SDPM has been discussed in, for example, “Spectral-domain phasemicroscopy” by M. A. Choma, A. K. Ellerbee, C. Yang, T. L. Creazzo, andJ. A. Izatt, Opt. Lett. 30(10), 1162 (2005) and “Spectral-domain opticalcoherence phase microscopy for quantitative phase-contrast imaging” byC. Joo, T. Akkin, B. Cense, B. H. Park, and J. F. DeBoer, Opt. Lett.30(16), 2131 (2005).

SUMMARY OF THE INVENTION

Some embodiments of the present invention provide optical coherencetomography systems including an OCT engine and a processor. The OCTengine is configured to provide both standard OCT imaging and spectraldomain phase microscopy (SDPM) imaging. The processor is coupled to theOCT engine and is configured to use a first signal processing methodwhen the OCT engine is configured to provide standard OCT imaging and asecond signal processing method when the OCT engine is configured toprovide SDPM imaging.

In further embodiments of the present invention, a scanning sample probemay be coupled to the OCT engine and may be configured to providelateral scanning of a sample and provide image information associatedwith the sample. In certain embodiments of the present invention, thescanning sample probe may include single mirrors, galvo mirror pairsand/or microelectromechanical mirrors.

In still further embodiments of the present invention, the imageinformation associated with the sample may include features in thesample larger than a resolution of from about 3.0 to about 10.0 μm.

Some embodiments of the present invention provide spectral domain phasemicroscopy (SDPM) systems including a transparent member and a scanningsample probe. The transparent member is positioned on a sample. A sampleprobe and a sample stage are configured to provide image informationthrough the transparent member of the sample such that image informationassociated with a first surface of the sample can be distinguished fromimage information associated with a second surface of the sample.

In further embodiments of the present invention, the sample probe may beconfigured to provide lateral scanning of the sample.

In still further embodiments of the present invention, the sample stagemay include a moveable sample stage. The sample may be positioned on themovable stage such that the moveable sample stage provides lateralscanning with respect to the sample probe.

In some embodiments of the present invention, one or more spacers may bepositioned between the transparent member and the scanning sample. Adistance provided by the one or more spacers between the sample and thetransparent member may be used to distinguish between the imageinformation associated with a first surface of the sample and the imageinformation associated with a second surface of the sample. The distancebetween the sample and the transparent member may be adjustable. Incertain embodiments of the present invention, the distance between thesample and the transparent member may be less than a thickness of thesample.

In further embodiments of the present invention, the distance betweenthe sample and the transparent member may be greater than a resolutionof the OCT-configured imaging system.

In still further embodiments of the present invention, the transparentmember may be a glass slide or a glass slide having an antireflectivecoating on at least one surface of the glass slide. The glass slide maybe optically flat to within a fraction of a wavelength of the imagingsystem. The uncoated glass slide may have a thickness of about 4.0 mm,about a depth window of the OCT imaging system or about at least thickerthan the sample.

In some embodiments of the present invention, the transparent member maybe an integral component of a sample structure. The transparent membermay be delineated from a remainder of the sample structure by a regularboundary observable with the OCT imaging system.

In further embodiments of the present invention, the system may furtherinclude an OCT engine and a processor. The OCT engine may be coupled tothe scanning sample probe and may be configured to provide both standardOCT imaging and SDPM imaging. The processor may be coupled to the OCTengine and configured to use a first signal processing method when theOCT engine is configured to provide standard OCT imaging and a secondsignal processing method when the OCT engine is configured to provideSDPM imaging.

Still further embodiments of the present invention provide OCT systemsincluding an OCT engine, a processor, a scanning sample probe, a samplestage and a transparent member. The OCT engine is configured to provideboth standard OCT imaging and spectral domain phase microscopy (SDPM)imaging. The processor is coupled to the OCT engine and configured touse a first signal processing method when the OCT engine is configuredto provide standard OCT imaging and a second signal processing methodwhen the OCT engine is configured to provide SDPM imaging. The scanningsample probe and the scanning sample stage are coupled to the OCTengine. The transparent member is positioned on a sample. The scanningsample probe and the scanning sample stage are configured to providelateral scanning of the sample through the transparent member andprovide image information associated with the sample such that imageinformation associated with a first surface of the sample can bedistinguished from image information associated with a second surface ofthe sample.

Some embodiments of the present invention provide OCT systems includingan OCT engine and a processor. The OCT engine is configured to provideboth standard OCT imaging and spectral domain phase microscopy (SDPM)imaging, obtain image data associated with a first pass through a sampleand obtain image data associated with a second pass through the sample.The processor is coupled to the OCT engine and configured to process theimage data associated with the first and second passes through thesample to determine a path length variation and provide an image havinga resolution based on the determined path length variation.

Further embodiments of the present invention provide methods ofprocessing OCT data. Both standard OCT imaging and spectral domain phasemicroscopy (SDPM) imaging are provided using a single OCT engine using afirst signal processing method when the OCT engine is configured toprovide standard OCT imaging and a second signal processing method whenthe OCT engine is configured to provide SDPM imaging.

In still further embodiments of the present invention, raw dataassociated with a sample from the OCT engine may be received at aprocessor. The DC background data may be subtracted from the raw data.Data remaining after the DC subtraction may be resampled to convert fromsampling in wavelength space to sampling in wavenumber space. Theresampled data may be Fourier transformed to transform wavenumber spaceto amplitude data and/or phase data. The second processing method mayinclude selecting phase data from a particular depth in the sample.Phase change and unwrapping operations may be performed with respect tothe selected phase data. The phase changed and unwrapped data may bescaled to provide an SDPM image.

In some embodiments of the present invention, the first processingmethod may include resealing the amplitude data to generate a standardOCT image.

Further embodiments of the present invention provide methods for imaginga sample and a transparent member positioned on a sample using SDPM.Image information is obtained from the sample when the sample is in afirst position and one or more surfaces of the transparent memberprovides one or more first interfaces for SDPM measurements of thesample in the first position and depths in the sample provide one ormore second interfaces for the SDPM measurements of the sample in thesecond position. Image information is obtained from the sample when thesample is in a second position. One or more surfaces of the transparentmember provides at one or more first interfaces for SDPM measurements ofthe sample in the second position and depths in the sample provide oneor more second interfaces for the SDPM measurements of the sample in thesecond position. The first position of the sample and the secondposition of the sample are distinguished based on the image informationobtained from the sample in the first and second positions.

In still further embodiments of the present invention, image informationmay be obtained from the sample in the first position by obtaining animage depicting spacing between the transparent member and the sample inthe first position, obtaining an image depicting a thickness of thesample in the first position and obtaining an image depicting a sum ofthe spacing between the transparent member and the sample in the firstposition and the thickness of the sample in the first position.

In some embodiments of the present invention, image information from thesample in the second position may be obtained by obtaining an imagedepicting spacing between the transparent member and the sample in thesecond position, obtaining an image depicting a thickness of the samplein the second position, and obtaining an image depicting a sum of thespacing between the transparent member and the sample in the secondposition and the thickness of the sample in the second position.

In further embodiments of the present invention, the obtained imagesfrom the sample in the first and second positions may be processed toprovide distinguishable images of the first and the second surfaces ofsample.

Still further embodiments of the present invention provide methods foracquiring high resolution SDPM by obtaining image data associated with afirst pass through a sample and obtaining image data associated with asecond pass through the sample. The image data associated with the firstand second passes through the sample are processed to determine a pathlength variation. An image having a resolution based on the determinedpath length variation is provided.

In some embodiments of the present invention, path length may bedirectly proportional to the resolution of the image.

Although embodiments of the present invention are primarily discussedabove with respect to systems and methods, related computer programproducts are also provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram illustrating conventional OpticalCoherence Tomography (OCT) imaging systems.

FIG. 2 is a schematic block diagram illustrating a conventional commonmode implementation of OCT imaging systems.

FIG. 3 is a schematic block diagram illustrating a Spectral Domain PhaseMicroscopy (SDPM) OCT imaging system according to some embodiments ofthe present invention.

FIG. 4 is a schematic block diagram illustrating an SDPM OCT imagingsystem according to some embodiments of the present invention.

FIG. 5 is a flowchart/diagram illustrating processing steps in thegeneration of SDPM images according to some embodiments of the presentinvention.

FIG. 6 is a schematic block diagram illustrating image symmetry in SDPMaccording to some embodiments of the present invention.

FIG. 7 is a schematic block diagram illustrating SDPM OCT imagingsystems including an antireflective slide and spacer according to someembodiments of the present invention.

FIG. 8 is a schematic block diagram illustrating SDPM OCT imagingsystems according to embodiments of the present invention illustrated inFIG. 7.

FIG. 9 is a schematic block diagram illustrating SDPM OCT imagingsystems according to embodiments of the present invention illustrated inFIG. 7.

FIG. 10 is a schematic block diagram illustrating SDPM OCT imagingsystems according to some embodiments of the present invention.

FIG. 11 is a schematic block diagram illustrating SDPM OCT imagingsystems according to embodiments of the present invention illustrated inFIG. 10.

FIG. 12 is a schematic block diagram illustrating SDPM OCT imagingsystems according to some embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention now will be described more fully hereinafter withreference to the accompanying drawings, in which illustrativeembodiments of the invention are shown. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.Like numbers refer to like elements throughout. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless expressly stated otherwise. Itwill be further understood that the terms “includes,” “comprises,”“including” and/or “comprising,” when used in this specification,specify the presence of stated features, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof. It will be understood thatwhen an element is referred to as being “connected” or “coupled” toanother element, it can be directly connected or coupled to the otherelement or intervening elements may be present. Furthermore, “connected”or “coupled” as used herein may include wirelessly connected or coupled.As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Some embodiments of the present invention will now be discussed withrespect to FIGS. 1 through 12. As illustrated therein, some embodimentsof the present invention consist of novel system designs for SpectralDomain Phase Microscopy (SDPM), which may provide standard OpticalCoherence Tomography (OCT) imaging and SDPM imaging simultaneously,enable specific identification of the location and/or orientation ofSDPM images, and/or increase the resolution of SDPM images as will bediscussed further herein.

Referring first to FIG. 1, a general diagram of an optical portion of aSpectral Domain-Optical Coherence Tomography (SD-OCT) system will bediscussed. As illustrated in FIG. 1, the SD-OCT system includes alow-coherence source 100, for example, a broadband light source,connected to an interferometer 101, which has a source arm 108, areference arm 106, a sample arm 107, and a spectrometer arm 103.Connected to the reference arm 106 is a reflective mirror 120, connectedto the sample arm 107 is a scanner 122, and connected to thespectrometer arm 103 is spectrometer 104. In some embodiments of thepresent invention, an optional isolator 105 may be provided in thesource arm 108 and one or more polarization controllers 102 may beprovided in the source arm 108, reference arm 106, and/or sample arm107. This system may be capable of acquiring OCT images of the sample121 with a resolution set by the bandwidth of the broadband source 100and typically in the range of from about 3.0 to about 20.0 μm.

Referring now to FIG. 2, like reference numerals refer to like elementsthroughout, thus, in the interest of brevity, details with respect tolike numbered elements of FIG. 1 will not be discussed further herein.As illustrated in FIG. 2, a common mode design may be used for aSpectral Domain-Optical Coherence Tomography (SD-OCT) system. In thecommon mode implementation, one or more surfaces in the sample 221 mayact as the reference reflector instead of having an independentreference arm. The example illustrated in FIG. 2 is a human cornea wherethe light reflected from the front of the cornea acts as the referencelight 211 and light reflected from other depths in the eye are thesample light 212. This technique typically works best where there is aclear interface that provides a reasonably strong reflection. Forexample, this technique may work well in eyes (human and animal), glassof any form, and plastics.

In some embodiments of the present invention, the reference reflectormay be outside of the sample. In these embodiments, the referencereflector is typically very close to sample. For example, a referencereflector outside the sample may be a partially reflective mirror thatis just in front of the sample or a coated fiber tip that is scannedover the surface of the sample.

It will be understood that although the example illustrated in FIG. 2 isa human cornea, embodiments of the present invention are not limited tothis configuration.

The common mode implementation may have some advantages overimplementations with separate sample and reference arms. Since thesample and reference light travel down the same path except for theextra path length that the sample light traverses in the sample, bothpaths may have very similar attenuation, polarization effects,dispersion effects, and environmental effects, such as thermal,vibrations, and the like.

SDPM takes advantage of this high level of stability between thereference and sample paths to generate image information based on thephase variation of the reference and sample light. By looking at changesin the phase over time or space, subresolution variations can bedetected, imaged, and measured. Instead of the from about 3.0 to about10.0 μm resolution images, SDPM typically can measure changes with aresolution of about 1.0 nm or better. SDPM is not imaging features, butrather is measuring changes in relative path lengths either over time orspace. This technique may be well suited to the SD-OCT and SS-OCTsystems due to their intrinsic phase stability and the fact that theycan be used in common mode system architectures.

It will also be understood that SDPM can be used with Time Domain OCT(TD-OCT) systems. Implementation in TD-OCT may be difficult due to thelarge amount of phase noise due to the reference arm changing length andthe fact that common mode implementations are difficult. SDPM may alsobe used with any of the Full Field OCT implementations and particularlywith the Full Field OCT implementations that rely on a Swept Sourcearchitecture without departing from the scope of the present invention.In Full Field OCT, the system typically has a two dimensional camera,which images some portion of the sample. A swept source laser may beused to generate an A-scan at each pixel on the camera. By comparingadjacent pixels, the same phase variation data can be generated that iscreated by sweeping the beam over the sample in the SD-OCT and SS-OCTimplementations.

Referring now to FIG. 3, an exemplary implementation of an SDPM systemaccording to some embodiments of the present invention will bediscussed. The system illustrated in FIG. 3 includes, an OCT engine 302,for example, the OCT engine of FIG. 2, a computer 300 for control of thesystem and display of the acquired images and a sample probe 304 forscanning the light beam 305 over the sample 306. In some embodiments ofthe present invention, the sample may also be scanned underneath astationary sample probe. The sample illustrated in FIG. 3 is a glassplate 306 with surface imperfections on the bottom surface. By scanningthe beam over the sample 306 and measuring phase changes betweensuccessive A-scans, the sub-resolution variations in the bottom surfaceof the glass plate can be imaged and measured. As discussed above, theimaging resolution of the OCT system may be from about 3.0 to about 10.0μm, but the measurement resolution for variations in the surface may beless than about 1.0 nm.

Referring now to FIGS. 4, 5 and 6, OCT/SDPM imaging systems thatcollect, process and display the data to provide both OCT imaging andSDPM imaging from the same system using the same raw data according tosome embodiments of the present invention will be discussed. The systemincludes a computer 400 or other data processing device coupled to anOCT engine 402 by a communications channel 401. The OCT engine 402 isfurther coupled to a scanning sample probe 404 by an optical fiber 403.It will be understood that although the connection 403 is discussedherein as being an optical fiber, the connection 403 can be provided byother means than optical means without departing from the scope of thepresent invention.

The sample probe 404 provides the lateral scanning of the system and mayuse any scanning system including single mirrors, galvo mirror pairs,MEMS mirrors and/or other beam deflection techniques. The beam 405 scansover the sample and provides image information on both features largerthan the OCT resolution, such as the inclusion 450 in the sample 406 andsub-resolution variations within the sample 406, such as the surfacevariations on the bottom side of the sample 406.

It will be understood that in some embodiments of the present invention,the OCT and SDPM systems may alternatively be configured to provide forlateral scanning of the sample under a stationary sample probe. Thisarrangement for obtaining lateral information about the sample isequivalent to providing the scanning mechanism in the probe. Thus, asused herein “a scanning sample probe” may include a scanning samplemechanism without departing from the scope of the present invention.

Referring now to FIG. 5, processing steps for the dual OCT/SDPM systemaccording to some embodiments of the present invention will be discussedwith respect to the flowchart of block 500. As illustrated in block 500,after raw data is received from the optical portion of the OCT engine,the DC background is subtracted out (block 560), and the data may beresampled (block 561) to convert from sampling in wavelength space tosampling in wavenumber space. Resampling may be used for SD-OCT systems,but may not be needed in all cases including SS-OCT systems withintrinsic sampling in wavenumber. Furthermore, other processing stepsmay be included to deal with dispersion in the system or other issueswithout departing from the scope of the present invention.

The data may be Fast Fourier Transformed (FFTed) (block 562) totransform from wavenumber space to physical (or depth) space. The FFTmay provide both amplitude data and phase data as illustrated in FIG. 5.In standard OCT systems the amplitude data is further processed viaresealing (block 563) and other processing steps to generate an OCTimage (block 564). However, the dual OCT/SDPM systems according to someembodiments of the present invention may further process the phase datato generate a sub-resolution phase change image.

In particular, the phase data may be selected from a particular depth(block 565) in the sample, such as the bottom surface of sample 506, andfed through a phase change and phase unwrapping step (block 566). Theresult may be appropriately scaled (block 567) and turned into an SDPMimage (block 568) that can be displayed to the system user. A graphicalrepresentation of the process discussed with respect to block 500 isillustrated by images 501, 502, and 503, where 501 is the standard OCTimage for the sample 506, note that the surface variation is not visiblein this image since it is sub-resolution, 502 is the raw phase data forthe depth of the image corresponding to the bottom surface of the sample506, and 503 is the unwrapped and scaled data that shows thesub-resolution variation in the bottom surface of the sample 506. Someembodiments of the present invention include the generation of both OCTand SDPM images of the sample 506 for the system user.

Referring now to FIG. 6, consequences of the fact that the SDPM maymeasure a change in some part of the sample relative to anotherinterface will be discussed. The sample 206 can be imaged with eitherthe flat side (side #1) up or the rough side (side #2) up and in bothcases the SDPM image 600 may be the same. The SDPM measures a change orvariation in a path length in the sample, in this particular case thevariation in the distance from side #1 to side #2, and uses twointerfaces or surfaces to generate the path length. SDPM typicallycannot inherently distinguish which end of the path length is changing,only that there is some change. Changes in the middle of the path lengthmay also appear in the SDPM image and the location of those changestypically cannot be inherently located.

Referring now to FIGS. 7, 8, and 9, some embodiments of the presentinvention that provide a mechanism for addressing the problem ofdistinguishing which interface in SDPM is changing or if both interfacesare changing at the same time will be discussed. Like reference numeralsrefer to like elements throughout, thus, details with respect toelements already discussed herein will not be repeated in the interestof brevity. In particular, the system as illustrated in FIG. 7 issimilar to the system discussed above with respect to FIGS. 4 through 6,but also includes a transparent member, for example, a glass slide 750on the sample 706. In some embodiments of the present invention, theglass slide 750 and the sample 706 may be separated by one or morespacers 751. It will be understood that the spacer 751 is optional andis provided for exemplary purposes only. The glass slide 750 may haveone or more sides that are anti-reflection (AR) coated and may bepositioned on any surface of the sample 706.

Referring now to FIG. 8, the effect of the AR coated glass slide 850 onthe SDPM image of the sample 806 will be discussed. The surface of theglass slide 850 that is not AR coated may provide one of the interfacesfor the SDPM measurements and various depths in the sample may providethe second interface for each of the SDPM measurements. The top portionof FIG. 8 illustrates the SDPM image 870 generated if the “rough” side(side #2) is up and the bottom portion shows the SDPM image 871generated if the “rough” side (side #2) is down. In some embodiments ofthe present invention, the spacing between the glass slide 850 and thesample 806 is less than the thickness of the sample and the surfaceflatness of the non-AR coated side of the glass slide 850 may be known.It will be understood by those having skill in the art that processingmay be simplified if the surface of the glass slide 850 is substantiallyflat.

As illustrated in the image 870, the top portion where the “rough” side(side #2) of the sample 806 is up generates multiple lines with SDPMdata including one at a depth of Δ1, one at Δ2, and one at Δ1+Δ2, whereΔ1 is the spacing between the glass slide and the sample and Δ2 is thethickness of the sample. The SDPM image from depth Δ1 gives thevariation of the bottom surface of the glass slide relative to the topsurface of the sample. The SDPM image from depth Δ2 gives the variationof the top surface of the sample relative to the bottom surface of thesample. The SDPM image from depth Δ1+Δ2 gives the variation of thebottom surface of the glass slide 850 relative to the bottom surface ofthe sample 806. It will be understood that the variation in the topsurface of the sample 806 may show up in the SDPM data at Δ1+Δ2, eventhough the top surface is not one of the interfaces involved. This maybe due to the fact that the SDPM is a measure of the variation in pathlength between two interfaces and may include all variation in betweenthe two interfaces. Thus, all three depths may return the same SDPM datashowing the variation in the top of the sample.

As illustrated in the image 871, in the bottom of FIG. 8 the sample isturned upside down and the smooth surface is now on top. Again multipleSDPM images can be generated. Looking at Δ1, Δ2, and one at Δ1+Δ2, thereis now a flat line at Δ1 as the variation in the bottom of the sample,where the “rough” surface (side #2) now is, is only present at Δ2 and atΔ1+Δ2.

Referring now to FIG. 9, processing steps in accordance with someembodiments of the present invention will be discussed. To complete thepicture the sample 906 now has variation in the both the top surface(side #1) and the bottom surface (side #2). Again assume that Δ1 is lessthan Δ2, although that is not a requirement. Graph 900 illustrates theSDPM data, phase change at each depth, graph 901 illustrates the topsurface of the sample, and graph 902 illustrates the bottom surface ofthe sample. SDPM data may be generated at multiple depths, the data fromΔ1, Δ2, and Δ1+Δ2 is illustrated in graph 900. The variation in the topsurface of the sample is just the data at depth Δ1 as illustrated ingraph 901. The SDPM data at Δ2, and Δ1+Δ2 include variations in both thetop and the bottom of the sample as illustrated in image 902. Since thevariation in the top surface is now known, it can be subtracted out toleave the variation in the bottom surface. Either data from Δ2 or Δ1+Δ2may work, for illustration Δ1+Δ2 is chosen and the data from Δ1 issubtracted leaving just the variation in the bottom surface of thesample. Thus, it may now be possible to generate SDPM images of the topsurface (side #1) and the bottom surface (side #2) of the sample 906without knowing in advance which side is which, i.e., the sample can beflipped over and accurate images of the top and bottom surface can stillbe generated.

According to some embodiments of the present invention illustrated inFIGS. 7 through 9, a known interface may be provided that is external tothe sample and breaks the intrinsic symmetry in the sample. Once thevariation in one surface in the sample is known, it is possible tocalculate the variation in other samples by subtracting out knownvariations to provide data on a new interface. It will be understoodthat although techniques discussed herein are illustrated with respectto two surfaces in the sample, embodiments of the present invention arenot limited to this configuration. For example, three or more interfacesmay be provided in the sample without departing from the scope of thepresent invention. As illustrated, there is little to no variation inthe middle of the sample, however, this technique may work forvariations in the middle of the sample provided that there is sufficientsignal to generate SDPM data at that particular depth in the sample.

Referring now to FIGS. 10 and 11, some embodiments of the presentinvention having multiple external interfaces will be discussed. Likereference numerals refer to like elements throughout, thus, elementsdiscussed above may not be discussed further herein in the interest ofbrevity. As an illustrative example, consider FIGS. 10 and 11, where aglass slide 1050/1150 having no AR coating is used. Again the glassslide 1050/1150 can be placed on any surface of the sample, asillustrated it is shown above the sample 1006/1106. It will beunderstood that although embodiments of the present inventionillustrated in FIGS. 10 and 11 do not include a spacer between thesample 1006/1106 and the glass slide 1050/1150, embodiments of thepresent invention are not limited to this configuration. A spacer can beused as discussed above without departing from the scope of the presentinvention.

As illustrated in FIG. 11, both sides (sides #3 and #4) of the glassslide 1150 can act as interfaces, thus, there are more depths in the OCTimage that may provide SDPM data and images. Assume for illustrationthat the thickness of the sample 1106 is Δ1, the gap between the sampleand the glass slide is Δ2, the thickness of the glass slide is Δ3 andthat Δ2<Δ3<Δ1. This technique may work for SDPM data generated atnumerous depths including Δ2, Δ3, Δ1, (Δ3+Δ2), (Δ1+Δ2), and (Δ1+Δ2+Δ3)as shown in the graph 1100. This SDPM data may be generated by foursides numbered #1, #2, #3, and #4, assuming no variations in between thesides. Table 1101 shows which SDPM data sets contain information abouteach surface. For example, the SDPM data from depth Δ1+Δ2 may haveinformation about side #1, side #2, and side #4. Box 1102 shows thecalculations that may be used according to some embodiments of thepresent invention to extract information about a particular side fromthe SDPM data generated. For example, the variation in side #1 can becalculated by taking the SDPM data from depth (Δ3+Δ2) and subtractingdepth Δ3.

There are multiple ways to calculate the SDPM data for some surfaces,for example, SDPM data for side #2 can be calculated from either(Δ1+Δ2)−(Δ2) or (Δ1+Δ2+Δ3)−(Δ3+Δ2). Note that this technique may workeven if Δ2 is very small, as in the case where the glass slide istouching the sample. Provided the variation in side #4 is known, thevariation in side #1 and side #2 can still be determined from SDPM datafrom Δ3, which now contains information about side #1, and data from(Δ1+Δ2+Δ3)−(Δ3), which provides the information about side #2.

Referring now to FIG. 12, a technique for acquiring higher resolutionSDPM images from a sample 1206 will be discussed. Embodiments of thepresent invention discussed above have primarily been for the case wherethe sample light makes a single roundtrip 1251 through the sample as isillustrated in the upper left in diagram #1. OCT images may also begenerated by light that makes multiple roundtrips through the sample asillustrated by the light path 1252 illustrated in diagram #2. Thestandard OCT image 1200 contains image data from the single pass case,the double pass case, and possibly others. Typically the SDPM image isgenerated from the single pass case as is shown in 1201 and 1203.However, the technique may work for sample light that takes any numberof roundtrips, as illustrated in the double pass case in 1202 and 1204.Since this light is reflected off of the “rough” surface twice insteadof once, it may experience twice as much path length variation and,thus, may generate SDPM images with twice the resolution. This may becountered by generally higher noise at deeper levels in the OCT and theinherent imaging depth limit of the OCT system.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. Therefore, it is to be understood that the foregoing isillustrative of the present invention and is not to be construed aslimited to the specific embodiments disclosed, and that modifications tothe disclosed embodiments, as well as other embodiments, are intended tobe included within the scope of the appended claims. The invention isdefined by the following claims, with equivalents of the claims to beincluded therein.

1. A spectral domain phase microscopy (SDPM) system, comprising: atransparent member positioned on a sample; a sample probe and a samplestage configured to provide image information through the transparentmember of the sample such that image information associated with a firstsurface of the sample is distinguished from image information associatedwith a second surface of the sample; and at least one spacer positionedbetween the transparent member and the scanning sample, wherein adistance provided by the at least one spacer between the sample and thetransparent member is used to distinguish between the image informationassociated with a first surface of the sample and the image informationassociated with a second surface of the sample.
 2. The system of claim1, wherein the sample probe is configured to provide lateral scanning ofthe sample.
 3. The system of claim 1, wherein the sample stage comprisesa moveable sample stage and wherein the sample is positioned on themovable stage such that the moveable sample stage provides lateralscanning with respect to the sample probe.
 4. The system of claim 1,wherein the distance between the sample and the transparent member isadjustable.
 5. The system of claim 1, wherein the distance between thesample and the transparent member is less than a thickness of thesample.
 6. The system of claim 1, wherein the distance between thesample and the transparent member is greater than a resolution of theOCT-configured imaging system.
 7. The system of claim 1, wherein thetransparent member comprises a glass slide or a glass slide having anantireflective coating on at least one surface of the glass slide. 8.The system of claim 7, wherein the glass slide is optically flat towithin a fraction of a wavelength of the imaging system.
 9. The systemof claim 8, wherein the uncoated glass slide has a thickness of about4.0 mm, about a depth window of the OCT imaging system or about at leastthicker than the sample.
 10. The system of claim 1, wherein thetransparent member is an integral component of a sample structure. 11.The system of claim 10, wherein the transparent member is delineatedfrom a remainder of the sample structure by a regular boundaryobservable with the OCT imaging system.
 12. The system of claim 1,further comprising: an OCT engine coupled to the scanning sample probeand configured to provide both standard OCT imaging and SDPM imaging;and a processor coupled to the OCT engine and configured to use a firstsignal processing method when the OCT engine is configured to providestandard OCT imaging and a second signal processing method when the OCTengine is configured to provide SDPM imaging.
 13. A method for imaging asample and a transparent member positioned on a sample using spectraldomain phase microscopy (SDPM), comprising: obtaining image informationfrom the sample when the sample is in a first position, wherein at leastone surface of the transparent member provides at least one firstinterface for SDPM measurements of the sample in the first position anddepths in the sample provide at least one second interface for the SDPMmeasurements of the sample in a second position; obtaining imageinformation from the sample when the sample is in a second position,wherein at least one surface of the transparent member provides at leastone first interface for SDPM measurements of the sample in the secondposition and depths in the sample provide at least one second interfacefor the SDPM measurements of the sample in the second position; anddistinguishing the first position of the sample and the second positionof the sample based on the image information obtained from the sample inthe first and second positions, wherein obtaining image information fromthe sample in the first position comprises: obtaining an image depictingspacing between the transparent member and the sample in the firstposition; obtaining an image depicting a thickness of the sample in thefirst position; and obtaining an image depicting a sum of the spacingbetween the transparent member and the sample in the first position andthe thickness of the sample in the first position.
 14. The method ofclaim 13, wherein obtaining image information from the sample in thesecond position comprises: obtaining an image depicting spacing betweenthe transparent member and the sample in the second position; obtainingan image depicting a thickness of the sample in the second position; andobtaining an image depicting a sum of the spacing between thetransparent member and the sample in the second position and thethickness of the sample in the second position.
 15. The method of claim14, wherein distinguishing further comprises processing the obtainedimages from the sample in the first and second positions to providedistinguishable images of the first and the second surfaces of sample.16. A computer program product for imaging a sample and a transparentmember positioned on a sample using spectral domain phase microscopy(SDPM), comprising: computer readable storage medium having computerreadable program code embodied in the medium, the computer readableprogram code comprising: computer readable program code configured toobtain image information from the sample when the sample is in a firstposition, wherein at least one surface of the transparent memberprovides at least one first interface for SDPM measurements of thesample in the first position and depths in the sample provide at leastone second interface for the SDPM measurements of the sample in a secondposition; computer readable program code configured to obtain imageinformation from the sample when the sample is in a second position,wherein at least one surface of the transparent member provides at leastone first interface for SDPM measurements of the sample in the secondposition and depths in the sample provide at least one second interfacefor the SDPM measurements of the sample in the second position; andcomputer readable program code configured to distinguish a first surfaceof the sample from a second surface of the sample based on the imageinformation obtained from the sample in the first and second positions,wherein the computer readable program code configured to obtain imageinformation from the sample in the first position comprises: computerreadable program code configured to obtain an image depicting spacingbetween the transparent member and the sample in the first position;computer readable program code configured to obtain an image depicting athickness of the sample in the first position; and computer readableprogram code configured to obtain an image depicting a sum of thespacing between the transparent member and the sample in the firstposition and the thickness of the sample in the first position.
 17. Thecomputer program product of claim 16, wherein the computer readableprogram code configured to obtain image information from the sample inthe second position comprises: computer readable program code configuredto obtain an image depicting spacing between the transparent member andthe sample in the second position; computer readable program codeconfigured to obtain an image depicting a thickness of the sample in thesecond position; and computer readable program code configured to obtainan image depicting a sum of the spacing between the transparent memberand the sample in the second position and the thickness of the sample inthe second position.
 18. The computer program product of claim 17,wherein the computer readable program code configured to distinguishfurther comprises computer readable program code configured to processthe obtained images from the sample in the first and second positions toprovide distinguishable images of the first and the second surfaces ofsample.